Retrovirus Transduction Kit

Catalog No. ABIN2344941

Product Details

Application: Transfected Cell Culture (TCC)

Brand: ViraDuctin™

Sample Type: Cell Extracts, Cell Samples

Characteristics: The ViraDuctin™-retrovirus complexes will also quickly sediment onto target cells resulting in fast delivery of the viruses than simple diffusion. Both removal of transduction inhibitors and quick delivery of viruses to cells result in higher gene transfer. ViraDuctin™ Retrovirus Transduction Kit provides the following advantages: • Higher transduction efficiency in many cell types compared to reagents such as Polybrene®. • Rapid purification and removal of transduction inhibitors. • Quick delivery of viruses to cells. • Ideal for transduction of nonpermissive cells such as primary cells and stem cells.

Components:

1. ViraDuctin™ Retrovirus Transduction Reagent A (100X) : One sterile tube - 1.0 mL
2. ViraDuctin™ Retrovirus Transduction Reagent B (100X) : One sterile tube - 1.0 mL
3. ViraDuctin™ Retrovirus Transduction Reagent C (8X) : One sterile bottle - 15 mL

Material not included:

1. Retroviral Stock Solution
2. Transfection Reagent
3. Cells and cell culture growth medium
4. 37 °C Incubator

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Proprietary reagent cocktail forms supercomplex with retrovirus
• Increases retrovirus transduction efficiency 2- to 6-fold compared to Polybrene®
• Useful for infection of non-permissive cells, including many primary cells and stem cells

Protocol: The following transduction protocol is written for a 24-well format. Refer to the below table for the appropriate dispensing volumes of other plate formats. Culture Dish 96-well 24-well 12-well 6-well 60-mm 10-cm Retrovirus/Culture 100 500 1000 2000 5000 10000 Media (μL) Reagent A (100X) 1 5 10 20 50 100 (μL) Reagent B (100X) 1 5 10 20 50 100 (μL) Final Transduction 102 510 1020 2040 5100 10200 Volume (μL) Reagent C (1X) 100 500 1000 2000 5000 10000 (μL) Table 1: Dispensing Volumes of Different Plate Formats 3 I. Transduction of Adherent Cells

1. The day before transduction, trypsinize and count the cells, plating 0.2-2 x 105 cells in 0.5 mL complete culture medium per well of a 24-well plate. Incubate cells at 37 °C overnight.
2. On the day of transduction, transfer 0.5 mL of your retroviral supernatant in a sterile tube. Add 5 μL of ViraDuctin™ Retrovirus Transduction Reagent A (100X) and mix by inverting. Immediately add 5 μL of ViraDuctin™ Retrovirus Transduction Reagent B (100X) and mix by inverting.
3. Incubate 30 minutes at 37 °C.
4. Centrifuge for 10 minutes at 12,000 g. Carefully remove and discard supernatant, resuspend the pellet in 0.5 mL of fresh culture medium used for your target cells.
5. Remove culture medium from the 24-well plate and apply all 0.5 mL of the retrovirus- ViraDuctin™ mixture to cells.
6. Incubate at 37 °C overnight.
7. Dilute the appropriate amount of ViraDuctin™ Retrovirus Transduction Reagent C (8X) to 1X with complete culture medium (for example, add 70 μL of 8X Reagent C to 490 μL of complete culture medium).
8. Remove the media containing Retrovirus-ViraDuctin™ complexes and replace with 0.5 mLof complete culture medium.
9. To completely remove the transduction complex, remove the culture medium and replace with 500 μL of the diluted ViraDuctin™ Retrovirus Transduction Reagent C (1X) in each well, gently rock the plate for 30-60 seconds. IMMEDIATELY aspirate the medium containing ViraDuctin™ Retrovirus Transduction Reagent C and replace with 0.5 mLof complete culture medium. Wash twice with complete culture medium to remove any residue complex.
10. 48-72 hrs after transduction, proceed with desired method of detection. To select stable cell clones, replace medium with fresh medium containing antibiotic every 3-4 days until antibiotic-resistant colonies can be identified. II. Transduction of Suspension Cells
    1. The day before transduction, trypsinize and count the cells, plating 0.2-2 x 105 cells in 0.5 mL complete culture medium per well of a 24-well plate. Incubate cells at 37 °C overnight.
    2. On the day of transduction, transfer 0.5 mL of your retroviral supernatant in a sterile tube. Add 5 μL of ViraDuctin™ Retrovirus Transduction Reagent A (100X) and mix by inverting. Immediately add 5 μL of ViraDuctin™ Retrovirus Transduction Reagent B (100X) and mix by inverting.
    3. Incubate 30 minutes at 37 °C.
    4. Centrifuge for 10 minutes at 12,000 g. Carefully remove and discard supernatant, resuspend the pellet in 0.5 mL of fresh culture medium used for your target cells.
    5. Pellet your suspension cells for 5 minutes at 1000 g. Remove supernatant and resuspend pellet in 0.5 mL of the medium containing retrovirus from step
    6. 6. Incubate at 37 °C overnight.
    7. Dilute the appropriate amount of ViraDuctin™ Retrovirus Transduction Reagent C (8X) to 1X with complete culture medium (for example, add 70 μL of 8X Reagent C to 490 μL of complete culture medium). 4
    8. Centrifuge cells for 5 minutes at 1000 g and remove supernatant. Resuspend pellet in 0.5 mL of Reagent C/medium mixture from step
    9. 9. IMMEDIATELY centrifuge for 5 minutes at 1000 g. Aspirate the supernatant containing Reagent C and replace with 0.5 mLof complete culture medium. Repeat twice to remove any residue complex.
    10. 48-72 hrs after transduction, proceed with desired method of detection. To select stable cell clones, replace medium with fresh medium containing antibiotic every 3-4 days until antibiotic-resistant colonies can be identified. Recent Product Citations
        1. Miyoshi, N. et al. (2010). Defined factors induce reprogramming of gastrointestinal cancer cells. PNAS 107:40-45. Notice to Purchaser This product is sold for research and development purposes only and is not to be incorporated into products for resale without written permission from Cell Biolabs. The patented technology is covered by an exclusive license. By the use of this product you accept the terms and conditions of all applicable Limited Use Label Licenses. You may contact our Business Development department at busdev@cellbiolabs.com for information on sublicensing this technology.

Restrictions: For Research Use only

Handling

Precaution of Use: Remember that you will be working with samples containing infectious virus. Follow the recommended NIH guidelines for all materials containing BSL-2 organims.

Storage: 4 °C

Storage Comment: Store all kit components at 4°C until their expiration dates. DO NOT FREEZE.

References

Liu, Burd, Coopersmith, Ford: "Retrogenic ICOS Expression Increases Differentiation of KLRG-1hiCD127loCD8+ T Cells during Listeria Infection and Diminishes Recall Responses." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 196, Issue 3, pp. 1000-12, (2016) (PubMed).

Miyoshi, Ishii, Nagai, Hoshino, Mimori, Tanaka, Nagano, Sekimoto, Doki, Mori: "Defined factors induce reprogramming of gastrointestinal cancer cells." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, Issue 1, pp. 40-5, (2010) (PubMed).

Target Details

Background: Retroviral gene transfer is a technique for efficiently introducing stable, heritable genetic material into the genome of any dividing cell type. Replication-incompetent retrovirus is usually produced through transfection of the retroviral vector into a packaging cell line. Retroviruses are classified according to the receptors used to enter host cells. Ecotropic virus can recognize a receptor found on only mouse and rat cells. Amphotropic virus recognizes a receptor found on a broad range of mammalian cell types. Retrovirus usually has lower titer than lentivirus which leads to poor gene transfer. The rate at which retroviral vectors bind to and infect cells is mostly controlled by diffusion. During retrovirus infection, only a small fraction of the retroviral vectors can transduce target cells. Virion adsorption is the limiting step of this process. Another cause of poor gene transfer of retrovirus is the presence of transduction inhibitors in retroviral supernatants such as proteoglycans and glycosaminoglycans. The use of polycations, such as Polybrene®, is standard in many retroviral infection protocols owing to the observations of improved infection efficiency by neutralizing the electrostatic repulsion between virion and cell membranes, but the transduction inhibitors still remain in the retroviral supernatant during infection. ViraDuctin™ Retrovirus Transduction Kit is a proprietary formulation for the rapid purification of retrovirus from inhibitors of transductions.